This invention relates to a plate useful in matrix-assisted laser desorption ionization (MALDI) mass spectrometry analysis and to processes for making and using the plate. More particularly, this invention relates to a MALDI plate having a hydrophobic surface and to processes for making and using the plate.
For the analysis of large molecules such as DNA, peptides, proteins and other biomolecules, mass spectrometry with MALDI ionization is a standard method. For the most part, time-of-flight mass spectrometers (TOF-MS) are used for this purpose, but ion cyclotron resonance spectrometers or Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers as well as high-frequency quadrupole ion trap mass spectrometers, and hybrid quadrupole time of flight mass spectrometers (Q-TOF) are all applicable for these applications. Normally, biomolecules are in an aqueous solution, but is not uncommon for these important building blocks to be dissolved in solutions that contain varying levels of organic solvents (such as acetonitrile), particularly when reversed phase chromatography is used for isolation and fractionation of complex mixtures of these molecules. The large or high molecular weight substances including the biosubstances, and biomolecules mentioned above, the molecules of which are to be analyzed, are often referred to as “analytes”.
The terms biomolecules or biosubstances here denote oligonucleotides, peptides and proteins (i.e., the essential building blocks of the living world) including their particular analogs and conjugates, such as glycoproteins or lipoproteins. In preparation for mass analysis, analytes are isolated from a biological source, including biological fluid (e.g., urine, bile or mucus, etc.), tissue, organ, cell line, etc., by various methods known to the artisan. Usually, cell lysis is performed, with soluble and insoluble fractions isolated by centrifugation. Often the soluble protein fraction can be used without further manipulation. However, it may be useful to fractionate such complex mixtures by a variety of methods including specific isolation of a protein family or complex by separation techniques, such as immunoprecipitation, or immunoaffinity chromatography, one or two-dimensional gel electrophoresis, ion exchange chromatography, reversed phase chromatography or a combination of two or more of these techniques. When proteins are isolated they may be analyzed directly, or following digestion with chemical or enzyme reagents (e.g., cyanogen bromide, trypsin, chymotrypsin, lysine endopeptidase, glutamic acid endopeptidase, pepsin or any other suitable protein cleavage reagent). If peptide fragments are produced these may be isolated and fractionated by one skilled in the art. Briefly, various modes of chromatography (such as reversed phase, anion and/or cation exchange, hydrophilic chromatography, hydrophobic chromatography, displacement chromatography, capillary electrophoresis) or combinations of two or more modes can be used to isolate and fractionate complex peptide mixtures. Analytes (mixtures of peptides and/or proteins) and a matrix solution are deposited on a sample plate, usually made of an electrically conductive material (e.g., stainless steel) in preparation for mass analysis using MALDI.
The choice of a matrix substance for MALDI mass spectrometry (MALDI MS) analysis is dependent upon the type of biomolecules analyzed, with more than a hundred different matrix substances having become known in the field over the past several years. The task of the matrix substance is to separate the sample molecules from each other, to bond them to the sample support plate, to transform them into the gas phase during laser bombardment by the formation of a vapor cloud without destroying the biomolecules and finally to ionize the sample molecules by protonation or deprotonation. It has been found advantageous to incorporate analyte molecules in some form into the usually crystalline matrix substances during their crystallization or at least into the boundary surfaces between the small crystals.
Various methods are known for applying the sample and matrix to a sample plate. The simplest of these involves pipetting a droplet of a solution with sample and matrix onto a clean, metal (e.g., stainless steel) sample support plate. This droplet wets an area on the metal surface, the size of which corresponds approximately to the diameter of the droplet and is dependent on the hydrophobic properties of the metal surface and the characteristics of the droplet. After the solution dries, the sample spot consists of small matrix crystals spread over the formerly wet area, whereby generally there is no uniform coating of the previously wetted area. In aqueous solutions, most of the small crystals of the matrix generally begin to grow at the periphery of the wetted area on the metal plate, growing toward the inside of the wetted area.
In high throughput MALDI MS analysis utilizing robotics to transfer and deposit samples at high rates of sample processing, it is important that the sample plates used in the processing have uniform surfaces on a plate by plate basis so as to provide improved reliability of the measured data. For high throughput processing and automated data collection, it is also important that the footprint area of the deposited samples for a fixed volume be uniform, small and predictable. The provision of a hydrophobic surface on a sample plate permits depositing samples having a smaller area and larger volume as compared to a metal sample plate having a nonhydrophobic surface. Additionally, the hydrophobic surface greatly minimizes the spread of liquid across the surface, thus avoiding cross-contamination of analyte samples. However, the plate surface should not be so hydrophobic to cause the contact angle of the deposited liquid sample to be exceedingly high thereby reducing the footprint area of the deposited sample. Such area reduction is undesirable since the laser subsequently used to vaporize the sample has an increased probability of striking the sample plate rather than the sample during automated operation. This is undesirable particularly in tandem mass spectroscopy (MS/MS) processes, which require relatively large samples, which, in turn, require 10,000 to 100,000 or more exposures of the sample to the laser (shots).
It has been proposed in U.S. Pat. No. 6,287,872 to coat the sample plate (usually made of stainless steel) with a hydrophobic coating of a fluorinated polymer such as polytetrafluoroethylene (TeflonR). While this coating provides a highly reproducible surface for sample and matrix depositions, such a polymer coating exhibits certain drawbacks. It has been found that the fluorinated polymer coating is evaporated essentially from the onset of exposing the samples to a laser thereby creating a neutral cloud, which in rapid fashion is deposited on the ion optical elements of the mass spectrometer used in the MALDI analysis. This contamination of the mass spectrometer causes it to become unstable, and constant retuning of instrument optics is required to maintain performance. Ultimately, such rapid coating of mass spectrometer ion optical elements, diminishes the effectiveness of the mass spectrometer to a level that performance can only be restored by cleaning the instrument. In addition, these coatings are relatively thick and therefore are not uniform. Furthermore, the fluorinated polymer coating is not removable from the sample plate under benign conditions so that it produces even more non-uniform results over repeated use in a MALDI process.
Hung et al., proposed (Anal. Chem., 1998, Vol. 70, N: 14, pp. 3088-3093) the use of a film of paraffin wax (referred to as Parafilm) applied over the metal probe to provide a hydrophobic surface for the sample probe tip. The Parafilm was first stretched to reduce its thickness and attached to the metal probe tip without using an adhesive to form a non-integral layer on the surface of the probe tip. As disclosed by Hung et al., a variation in peak position was observed from sample to sample, which could be caused by an uneven coating surface level. The non-uniformity is primarily due to the fact that the coating obtained with stretched Parafilm is too thick to permit control of surface uniformity, which is compounded by the non-integral attachment of the Parafilm. Providing a uniform sample surface is a key parameter allowing reliable reuse of the sample plate.
Accordingly, it would be desirable to provide a sample plate for use in a MALDI MS process, which has a uniform, easily removed hydrophobic surface that is reproducible from plate to plate. Such a sample plate would permit accurate positioning of samples on the plate in a repetitive manner so that the plate can be reused many times. Additionally, the coating would be stable, and not volatilized by the ionization process, thereby limiting its contribution to instrument contamination.